Effect of Complexes of ADP and Phosphate Analogs on the Conformation of the Cys707‐Cys697 Region of Myosin Subfragment 1

Phan, Brigitte C.; Peyser, Y. Michael; Reisler, Emil; Mühlrád, András

doi:10.1111/j.1432-1033.1997.00636.x

Cited by 36 publications

(50 citation statements)

References 45 publications

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“…However, fast-reacting cysteines against DTNB per mole of S1 are largely oxidized (1.5 over 2, i.e., 75%) during treatments with SIN-1 producing 60% inhibition of S1 ATPase activities, and it has been shown that oxidation or chemical modification of the two highly reactive cysteines of S1, Cys 707 and Cys 697 , results in strong inhibition of the S1 ATPase activities (28,45,51,52). This point was confirmed by the tight correlation between the fractional decrease of the extent of labeling of Cys 707 with IAF and the fractional decrease of S1 ATPase activity after exposure to SIN-1 reported in this article, as ascertained by S1 Cys 707 titration with IAF under well-established experimental conditions (34,35,45). These cysteines are located in the catalytic domain of S1, very close to each other in the 3D structure of a domain that undergoes significant conformational changes during the catalytic cycle (53,54), and the DSC results reported in this article showed that SIN-1-treated S1 has a largely impaired ability to attain the conformation of the obligatory intermediate catalytic state S1‚Mg 2+ -ADP‚P i , which in nontreated S1 is induced upon incubation with Mg 2+ -ADP‚V 1 (31,32).…”

Section: Discussionsupporting

confidence: 79%

“…This is in good agreement with the conclusions reached on the basis of Ca 2+ -ATPase and K + /EDTA-ATPase activity measurements (see above). Because S1 can be selectively labeled by IAF at Cys 707 (34,35,45), its oxidation by SIN-1 can also be monitored by the parallel decrease of the extent of labeling of S1 by IAF under the standard experimental conditions indicated in Materials and Methods. The results shown in Figure 3B fully confirmed this point and also demonstrated a tight correlation between the fractional decrease of the extent of labeling of Cys 707 with IAF and the fractional decrease of the S1 K + /EDTA-ATPase activity.…”

Section: Inhibition Of S1 Atpase By Sin-1 and Synthetic Peroxynitritementioning

confidence: 99%

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Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

et al. 2006

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Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg 2+ -ATPase activity, reaching 50% inhibition with 46.7 ( 8.3 µM SIN-1 for 8.7 µM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg 2+ -ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca 2+ -dependent and K + /EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg 2+ -ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys 707 and Cys 697 ), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5′-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys 707 labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg 2+ -ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP‚V 1 to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

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Section: Discussionsupporting

confidence: 79%

Section: Inhibition Of S1 Atpase By Sin-1 and Synthetic Peroxynitritementioning

confidence: 99%

Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

et al. 2006

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“…Supporting evidence lies in the fact that modification of the actin C-terminus by proteolysis, antibody binding and by mutagenesis can significantly impact actin polymerization properties [Johannes and Gallwitz, 1991;Crosbie et al, 1994;Orlova and Egelman, 1995;Phan et al 1997;Hegyi et al, 1998;Kim et al, 1998]. Attachment of fluorometric probes to different parts of the actin showed that polymerization per se and the binding of specific proteins to the actin filament at a distance from the probe site could affect probe behavior [Feng et al, 1997;Moens and dos Remedios, 1997;Wen et al, 2000;Bobkov et al, 2002Bobkov et al, , 2004Borovikov et al, 2004].…”

Section: Budding Yeast As An Expression System For Mutant Actinsmentioning

confidence: 99%

“…These interacting structural elements could constitute an allosteric system through which actin-binding proteins on the filament surface could affect filament formation and stability. For example, regulatory proteins such as Arp2/3 and formin which bind at or near K118 on the surface [Rouiller et al, 2008;Goley et al, 2010; Thompson et al, 2013] could initiate a set of allosteric conformational changes to the strand-strand interface or C-terminus known to be important in actin polymerization [Johannes and Gallwitz, 1991;Crosbie et al, 1994;Orlova and Egelman, 1995;Phan et al 1997;Hegyi et al, 1998; Kim et al, 1998a,b] affecting both filament formation and stability.They termed this helical element the pathogenic helix because disease-causing mutations cluster in it and the elements with which it interacts. R256H in a-smooth muscle actin causes TAAD.…”

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confidence: 99%

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confidence: 99%

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Insights into the effects of disease‐causing mutations in human actins

Rubenstein

Wen

2014

Cytoskeleton

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Mutations in all six actins in humans have now been shown to cause diseases. However, a number of factors have made it difficult to gain insight into how the changes in actin functions brought about by these pathogenic mutations result in the disease phenotype. These include the presence of multiple actins in the same cell, limited accessibility to pure mutant material, and complexities associated with the structures and their component cells that manifest the diseases. To try to circumvent these difficulties, investigators have turned to the use of model systems. This review describes these various approaches, the initial results obtained using them, and the insight they have provided into allosteric mechanisms that govern actin function. Although results so far have not explained a particular disease phenotype at the molecular level, they have provided valuable insight into actin function at the mechanistic level which can be utilized in the future to delineate the molecular bases of these different actinopathies. Key Words: actin; allostery; disease; mutation; isoforms Introduction A ctin mutations can cause human disease. However, little is known concerning the mechanisms by which these mutations lead to the disease phenotypes they produce. The goal of this review is to describe the general properties of actin, the diseases associated with mutations in actin, methodologies that might be employed to achieve a mechanistic understanding of actin based disease and efforts that have been made toward this goal. Properties of G-and F-ActinA discussion of the effects of pathogenic mutations on actin function requires a basic understanding of actin structure and its solution properties. Actin is an abundant 42 kD monomeric protein, found in virtually all eukaryotic cells, that can cycle between either a monomeric (G-actin) or polymeric (F-actin) state. It plays both contractile and structural roles in the cytoplasm, and recent studies have documented that actin in the nucleus acts as a promoter of transcription or as a member of a number of chromatin remodeling complexes Philimonenko et al., 2004;Miyamoto and Gurdon, 2011; Miyamoto et al., 2011a,b;Weston et al., 2012].G-actin is a clam-shaped protein with two domains, the inner and outer domains ( Fig. 1), connected by a hinge region, and separated by a deep cleft. Outer (subdomains 1 and 2) and inner (subdomains 3 and 4) refer to their position in the actin filament. The N and C-termini reside on opposite faces of the protein in subdomain 1. An adenine nucleotide binds deep within the inter-domain cleft, held in place by a divalent cation, and imparts stability to the protein and the filament. This latter role depends on its ability to cycle between the ATP and ADP state by virtue of a polymerization-dependent ATPase activity [Carlier et al., 1987;Korn et al., 1987]. F-actin is a polar structure with pointed (2) and barbed (1) ends referring to the arrowhead pattern formed when myosin S1 binds to F-actin in the absence of ATP [Craig et al., 1985]. The polarity of th...

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Functional Analysis of a De NovoACTBMutation in a Patient with Atypical Baraitser-Winter Syndrome

et al. 2013

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Exome sequence analysis can be instrumental in identifying the genetic etiology behind atypical disease. We report a patient presenting with microcephaly, dysmorphic features, and intellectual disability with a tentative diagnosis of Dubowitz syndrome. Exome analysis was performed on the patient and both parents. A de novo missense variant was identified in ACTB, c.349G>A, p.E117K. Recent work in Baraitser-Winter syndrome has identified ACTB and ACTG1 mutations in a cohort of individuals and we rediagnosed the patient with atypical Baraitser-Winter syndrome. We performed functional characterization of the variant actin and show that it alters cell adhesion and polymer formation supporting its role in disease. We present the clinical findings in the patient, comparison of this patient to other patients with ACTB/ACTG1 mutations, and results from actin functional studies that demonstrate novel functional attributes of this mutant protein.

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Effect of Complexes of ADP and Phosphate Analogs on the Conformation of the Cys707‐Cys697 Region of Myosin Subfragment 1

Cited by 36 publications

References 45 publications

Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

Insights into the effects of disease‐causing mutations in human actins

Functional Analysis of a De NovoACTBMutation in a Patient with Atypical Baraitser-Winter Syndrome

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